Separations-based analyses are a prominent part of biological research, allowing one to characterize different biological samples, reaction products and the like. Examples of some of the more prevalent separations-based analyses include electrophoretic separations of macromolecular species, e.g., proteins and nucleic acids. Electrophoresis, e.g., capillary electrophoresis, has been established as a highly effective method for separating macromolecular species in order that they might be further characterized. Protein and nucleic acid molecules are two major examples of molecular species that are routinely fractionated and characterized using electrophoretic systems.
Both microfluidic and macrofluidic devices have been applied in separations-based analyses. Examples of novel microfluidic devices and methods for use in the separation of molecular, and particularly macromolecular species by electrophoretic means are described in U.S. Pat. Nos. 5,958,694, 6,032,710, and 7,419,784, for example, the entire contents of which are incorporated by reference herein. In such devices, the sample containing the macromolecular species for which separation is desired is placed in one end of a separation channel located in a microfluidic substrate and a voltage gradient is applied along the length of the channel. As the sample components (also referred to as “fragments”) are electrophoretically transported along the length of the channel and through the separation (sieving) matrix disposed therein, those components are resolved. The separated components are then detected at a detection point along the length of the channel, typically near the terminus of the separation channel downstream from the point at which the sample was introduced. Following detection, the separated components are typically directed to a collection reservoir/well in the device (or to an external device such as a multiwell plate via a capillary pipettor, for example) for subsequent extraction or disposal.
In many situations, it is desirable to extract selected fragments of interest, such as DNA fragments, following the separation of the fragments into bands in the separation matrix for further processing or analysis, e.g., restriction enzyme modification, T4 ligation, PCR amplification, mass spectroscopy, or polynucleotide kinase reactions. The typical process used by laboratory researchers for extracting and isolating selected DNA fragments of interest (and other desired nucleic acid and protein fragments) from a separation matrix (such as an agarose gel) involves excising the desired fragments from the separation matrix and then extracting and purifying the excised fragment(s). First, the separated fragments are stained and illuminated by shining ultraviolet (UV) light on the fragments to visualize the separated bands. A razor blade is then used to manually cut above and below each fragment of interest so that one or more slices of the sieving can be removed. Then the DNA is extracted from the removed slices using various solutions and heating to dissolve the sieving matrix. The DNA can be further purified by standard solid phase extraction (binding the DNA to a solid surface such as glass followed by washing and finally elution). The recovered DNA can then be used for further processing or analysis. This extraction process, however, is time consuming, laborious and potentially damaging to the DNA (e.g., nicking of the DNA can occur if the DNA is exposed to ultraviolet light too long while the fragments of interest are being illuminated for excision).
Thus, in performing separations-based analyses, it would be desirable to be able to also isolate or extract one or more of the separated components in the device itself for further analysis or processing. The recovered or isolated fragments could then be used for a variety of different processes including, for example, the following: amplification using polymerase chain reaction (PCR); ligation reactions for cloning small to medium-sized strands of DNA into bacterial plasmids, bacteriophages, and small animal viruses to allow the production of pure DNA in sufficient quantities to allow its chemical analysis; adapter ligation used in high-throughput sequencing; reactions to dissolve a separated protein or nucleic acid component in a suitable matrix for further analysis by a mass spectrometer using, for example, Matrix-Assisted Laser Desorption Ionization (MALDI); binding reactions to bind a labeling agent to one or more separated protein or nucleic acid components for further analysis; or other similar post-detection processes. In addition, in the case of PCR samples, it is important to be able to separate smaller dimer and primer molecules from the main nucleic acid fragments in the sample and then isolate and collect the main nucleic acid fragments for further analysis or processing, while directing the smaller primer and dimer components to a waste reservoir/cell for removal and subsequent disposal.
A standard reference of known size is obtained by separating a standard DNA sizing ladder, e.g., for DNA separations, or a standard polypeptide of known molecular weight, e.g., for protein separations. Such a sizing ladder allows the size of unknown fragments to be determined. In a typical separation assay without fractionation (i.e., without isolation of components), the step of separating a standard sizing ladder is performed prior to transporting the first sample material through the separation channel to separate the sample material into a plurality of sample components. Thus, the entire ladder and all separated sample components have passed the detector before any sizing analysis is performed. (A ladder obtained from previous experiments is often stored and may be termed an archival ladder.) Alternatively, a ladder may be run in parallel with a sample (and may be termed a real-time ladder). In either case, the entire ladder and all separated sample components have passed a detector before any sizing analysis is performed.
To permit fractionation, a sizing analysis may need to be performed prior to the sample component(s) of interest passing the detector, thereby allowing a selected one or more separated components of interest to be diverted from the separation channel to a sample component collection location based on the determined size of the selected one or more sample components. I.e., a fractionation device may need to perform real-time sizing in order to determine the precise time to divert the material of interest to the sample component collection location. Thus, to improve sizing determinations in a fractionation device, it would be advantageous to provide methods of performing a sizing analysis using a corrected sizing ladder.